COL5A1 Exon 14 Splice Acceptor Mutation Causes a Functional Null Allele, Haploinsufficiency of α1(V) and Abnormal Heterotypic Interstitial Fibrils in Ehlers-Danlos Syndrome II

We studied four affected individuals from a family of three generations with Ehlers-Danlos Syndrome II. Type V collagen transcripts of affected individuals were screened by reverse transcriptase-polymerase chain reaction. Amplification of the exon 9–28 region of α1(V) yielded normal and larger products from the proband. Sequencing of cDNA revealed a 100-base pair insertion from the 3′-end of intron 13 between exons 13 and 14 in one allele. The genomic defect was identified as an A−2→ G substitution at the exon 14 splice acceptor site. A cryptic acceptor site −100 nucleotide within intron 13 is used instead of the mutant splice site. The insertion shifts the reading frame +1 and results in a stop codon within exon 17. The mutant transcript was much less abundant than normal allele product in untreated cultured fibroblasts but was approximately equimolar in cycloheximide-treated cells, suggesting that the mutation causes nonsense-mediated decay of mRNA. By RNase protection experiments, the level of mutant transcript was determined to be 8% that of the normal transcript in untreated proband fibroblasts. Relative to type I collagen, proband fibroblasts secreted only 65% of the amount of type V collagen secreted by normal controls. Selective salt precipitation of proband secreted collagen provided supportive evidence that the α chain composition of type V collagen remains α1(V)2α2(V) even in the context of α1(V) haploinsufficiency. Type V collagen incorporates into type I collagen fibrils in the extracellular matrix and is thought to regulate fibril diameter. Transmission electron micrographs of type I collagen fibrils in a proband dermal biopsy showed greater heterogeneity in fibril diameter than in a matched control. The proband had a greater proportion of both larger and smaller fibrils and occasional fibrils with a cauliflower configuration. Unlike the genotype/phenotype relationship seen for type I collagen defects and osteogenesis imperfecta, the null allele in this family appears to cause clinical features similar to those seen in cases with structural alterations in type V collagen. We studied four affected individuals from a family of three generations with Ehlers-Danlos Syndrome II. Type V collagen transcripts of affected individuals were screened by reverse transcriptase-polymerase chain reaction. Amplification of the exon 9–28 region of α1(V) yielded normal and larger products from the proband. Sequencing of cDNA revealed a 100-base pair insertion from the 3′-end of intron 13 between exons 13 and 14 in one allele. The genomic defect was identified as an A−2→ G substitution at the exon 14 splice acceptor site. A cryptic acceptor site −100 nucleotide within intron 13 is used instead of the mutant splice site. The insertion shifts the reading frame +1 and results in a stop codon within exon 17. The mutant transcript was much less abundant than normal allele product in untreated cultured fibroblasts but was approximately equimolar in cycloheximide-treated cells, suggesting that the mutation causes nonsense-mediated decay of mRNA. By RNase protection experiments, the level of mutant transcript was determined to be 8% that of the normal transcript in untreated proband fibroblasts. Relative to type I collagen, proband fibroblasts secreted only 65% of the amount of type V collagen secreted by normal controls. Selective salt precipitation of proband secreted collagen provided supportive evidence that the α chain composition of type V collagen remains α1(V)2α2(V) even in the context of α1(V) haploinsufficiency. Type V collagen incorporates into type I collagen fibrils in the extracellular matrix and is thought to regulate fibril diameter. Transmission electron micrographs of type I collagen fibrils in a proband dermal biopsy showed greater heterogeneity in fibril diameter than in a matched control. The proband had a greater proportion of both larger and smaller fibrils and occasional fibrils with a cauliflower configuration. Unlike the genotype/phenotype relationship seen for type I collagen defects and osteogenesis imperfecta, the null allele in this family appears to cause clinical features similar to those seen in cases with structural alterations in type V collagen. Ehlers-Danlos Syndrome (EDS)1 is a heterogeneous group of connective tissue disorders (1Beighton P. Beighton P. McKusick's Heritable Disorders of Connective Tissue.Mosby, Inc. 1992; : 189-251Google Scholar). Clinical manifestations in the milder forms of the disorder occur predominantly in the dermis (loose skin, abnormal scars, easy bruising) and joints (hyperextensibility), whereas the vascular (aneurysms) and visceral (ruptures, pneumothoraces) symptoms are found in the severe type IV form of EDS. A corresponding heterogeneous range of gene defects causes the broad range of EDS forms. These forms have involved the genes for type I (EDS VII) or III (EDS IV) collagen or for enzymes involved in the collagen metabolic pathway (EDS VI and VII) (2Steinmann B. Royce P.M. Superti-Furga A. Royce P.M. Steinmann B. Connective Tissue and Its Heritable Disorders.Wiley-Liss, Inc. 1993; : 351-408Google Scholar). Recently, a noncollagenous defect in tenascin-X has also been demonstrated in EDS (3Burridge S.M. Schalkwajk J. Taylor G. Steijlen P.M. Miller W.L. Bristow J. Am. J. Hum. Genet... 1999; 65 (Abstr. 1604): A286Google Scholar). Furthermore, there must be a number of additional genes in which defective forms cause EDS, because many EDS patients do not have mutations in the genes already described.In the last 4 years, type V collagen joined the growing list of matrix molecules associated with Ehlers-Danlos Syndrome. Type V collagen was a prime candidate for such a role because it is present as a minor component of the extracellular matrix in tissues in which type I collagen is the predominant structural molecule, especially skin and tendon (4Fessler L.I. Shigaki N. Fessler J.H. J. Biol. Chem... 1985; 260: 13286-13293Google Scholar). Type V collagen is a fibrillar collagen in which the central helical region is the same length as that of type I collagen. It occurs as two forms of heterotrimers or α1(V)3homotrimer. In the dermis, tendon, and bone, α1(V)2α2(V) is the usual trimer composition (5Burgeson R.E. Hebda P. Morris N. Hollister D. Proc. Natl. Acad. Sci. U. S. A... 1976; 257: 7852-7856Google Scholar, 6Jimenez S.A. Yankowski R. Bashey R.I. Biochem. Scholar, Tissue role of this type V collagen in the matrix appears to the of fibrils with type I collagen and a the diameter of those The of fibrils by I and V collagen was the of collagen into fibrils to be demonstrated J. Scholar). with was used to I and V collagen in and that type V were fibrils were fibrils are in and type V is abundant than in type I collagen predominant a role for type V was in the diameter of was of I and V collagen. the proportion of type V collagen in a of fibril diameter J. Scholar). The of type V α was to the the of type V at the of type I fibrils with the helical of type V and the of type V the and fibril by Fessler L.I. J. 1993; Scholar). role was by gene of in abnormal the have tissue and fibrils that are heterogeneous in R. Genet... structural defects and null of type V collagen have been in patients with EDS. patients have had EDS I or the and forms in the P. A. D. G. R. Hollister R.E. D. P. D. Am. J. Genet... also as the type in the P. A. Steinmann B. P. Am. J. Genet... Scholar). the structural defects in type V collagen, were in the α1(V) and three were in the chain Miller Genet... Scholar, Hum. Genet... Scholar, S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar, S. J. Genet... Scholar). cause two cause and one results from a Transmission electron micrographs of fibrils in four cases are by the of fibril and in diameter. null of have been as a cause of a proportion of EDS Steinmann B. Am. J. Hum. Genet... Am. J. Hum. Genet... Scholar). Transmission electron micrographs in one showed larger and fibrils Am. J. Hum. Genet... the present have and a type V collagen mutation a null allele. of type V collagen is both collagen and salt precipitation of proband secreted collagen of the α1(V)2α2(V) chain composition of type V collagen even in the context of α1(V) haploinsufficiency. dermal fibrils have greater heterogeneity of diameter than in the control. The of for the genotype/phenotype in EDS are a type V collagen defect in a with type of Ehlers-Danlos Syndrome. collagen exons an of the helical R. B. Proc. Natl. Acad. Sci. U. S. A... defects that in the of an exon the reading frame and cause the of a abnormal α the G defect in an α1(V) exon 14 acceptor site in the of a cryptic acceptor site −100 in intron The cryptic acceptor had the than the usual of α1(V) exon 14 and fibrillar collagen splice acceptor In there were a of in the 3′-end of intron 13 in which the from to of the mutant transcript by with an at the 3′-end of intron 13 with in exon or 13 revealed additional The of the −100 site from the of a or in the The site at with or was the candidate and have been in α1(V) chain be from this transcript with a the insertion shifts the collagen reading frame +1 and causes the of a stop codon in exon 17. α chain from the mutant transcript be into V the and region in the of α J. Scholar). In the α1(V) exon 14 the between the and the helical region Scholar). transcripts α helical a and be only a amount of even the chain be because the mutant transcript is to nonsense-mediated decay and is present in the fibroblasts at only 8% the level of normal These features to a null allele at the is in the of type V collagen secreted by the fibroblasts of the proband. the of secreted collagen is to of type I collagen as the proband as much type V collagen as the control. that the fibrils of the proband must be with a type I collagen Selective salt precipitation was to the of the type V trimer secreted in the context of α1(V) has the same of α1(V) and have been that the chain was a of the tissue In only α1(V) was Miller Scholar). In Miller and skin A. Biochem. the two were present in a In bone, the trimer was of a of and the chain Tissue Scholar). Furthermore, the chain is in fibroblasts J. Biol. Chem... Scholar). of α1(V) the of the type V In the collagen secreted by the type V collagen forms were at the and the α1(V) and were present in a is that trimer composition is tissue from an the type V collagen in proband is secreted in a amount and with the usual α chain the of this mutant for matrix and for must from V than a is a for a proportion of EDS I and of which this is the in and Am. J. Hum. Genet... α1(V) from cases of EDS I or In were to or or to stop Steinmann B. Am. J. Hum. Genet... the of EDS patients for of patients were for at one length and of had only one allele in The of two of patients that at of EDS I or patients have mutations in which results in haploinsufficiency. Furthermore, of mutations in of mutant transcript by nonsense-mediated decay is one type of which transcripts with are by a D. R. Scholar). are to have a greater decay in the transcript J. and the codon in this that must also be because of the cases of EDS with are in the The context of the codon is also to be the of one or by A. 1993; Scholar). are 14 and of the stop codon in the mutant transcript of this null mutation with V structural defects genotype/phenotype of V structural of α1(V) and three structural of have been Miller Genet... Scholar, Hum. Genet... Scholar, S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar, S. J. Genet... Scholar). are and the are or and a The of patients with V structural the range of EDS I II. The family has to EDS II. The cases in the by Am. J. Hum. Genet... and Steinmann B. Am. J. Hum. Genet... the EDS to type V appears to have a than a null allele of type I collagen. type I collagen, null of are for the form of osteogenesis whereas structural defects of type I collagen cause the severe to and IV of osteogenesis In EDS, the of to be similar to those of structural between the genotype/phenotype of I and V collagen in in type I collagen null a of fibrils not have a tissue and causes osteogenesis In because of the role of type V collagen in fibril both and structural defects of type V collagen cause of fibril which are to the of the skin and The four cases of EDS with V structural defects for which electron of fibrils has been defects of exon α1(V) exon α1(V) exon and an α1(V) substitution S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar). the same of fibril and in diameter. It is that this is also the fibril seen in this in which the defect causes because a in fibril diameter have been that the of fibril in is not the of V and I collagen. there is a in the of type V collagen fibrils with fibrils normal in and In of the of null mutations in and fibril a for to the of Ehlers-Danlos Syndrome (EDS)1 is a heterogeneous group of connective tissue disorders (1Beighton P. Beighton P. McKusick's Heritable Disorders of Connective Tissue.Mosby, Inc. 1992; : 189-251Google Scholar). Clinical manifestations in the milder forms of the disorder occur predominantly in the dermis (loose skin, abnormal scars, easy bruising) and joints (hyperextensibility), whereas the vascular (aneurysms) and visceral (ruptures, pneumothoraces) symptoms are found in the severe type IV form of EDS. A corresponding heterogeneous range of gene defects causes the broad range of EDS forms. These forms have involved the genes for type I (EDS VII) or III (EDS IV) collagen or for enzymes involved in the collagen metabolic pathway (EDS VI and VII) (2Steinmann B. Royce P.M. Superti-Furga A. Royce P.M. Steinmann B. Connective Tissue and Its Heritable Disorders.Wiley-Liss, Inc. 1993; : 351-408Google Scholar). Recently, a noncollagenous defect in tenascin-X has also been demonstrated in EDS (3Burridge S.M. Schalkwajk J. Taylor G. Steijlen P.M. Miller W.L. Bristow J. Am. J. Hum. Genet... 1999; 65 (Abstr. 1604): A286Google Scholar). Furthermore, there must be a number of additional genes in which defective forms cause EDS, because many EDS patients do not have mutations in the genes already In the last 4 years, type V collagen joined the growing list of matrix molecules associated with Ehlers-Danlos Syndrome. Type V collagen was a prime candidate for such a role because it is present as a minor component of the extracellular matrix in tissues in which type I collagen is the predominant structural molecule, especially skin and tendon (4Fessler L.I. Shigaki N. Fessler J.H. J. Biol. Chem... 1985; 260: 13286-13293Google Scholar). Type V collagen is a fibrillar collagen in which the central helical region is the same length as that of type I collagen. It occurs as two forms of heterotrimers or α1(V)3homotrimer. In the dermis, tendon, and bone, α1(V)2α2(V) is the usual trimer composition (5Burgeson R.E. Hebda P. Morris N. Hollister D. Proc. Natl. Acad. Sci. U. S. A... 1976; 257: 7852-7856Google Scholar, 6Jimenez S.A. Yankowski R. Bashey R.I. Biochem. Scholar, Tissue Scholar). The role of this type V collagen in the matrix appears to the of fibrils with type I collagen and a the diameter of those The of fibrils by I and V collagen was the of collagen into fibrils to be demonstrated J. Scholar). with was used to I and V collagen in and that type V were fibrils were fibrils are in and type V is abundant than in type I collagen predominant a role for type V was in the diameter of was of I and V collagen. the proportion of type V collagen in a of fibril diameter J. Scholar). The of type V α was to the the of type V at the of type I fibrils with the helical of type V and the of type V the and fibril by Fessler L.I. J. 1993; Scholar). role was by gene of in abnormal the have tissue and fibrils that are heterogeneous in R. Genet... Scholar). structural defects and null of type V collagen have been in patients with EDS. patients have had EDS I or the and forms in the P. A. D. G. R. Hollister R.E. D. P. D. Am. J. Genet... also as the type in the P. A. Steinmann B. P. Am. J. Genet... Scholar). the structural defects in type V collagen, were in the α1(V) and three were in the chain Miller Genet... Scholar, Hum. Genet... Scholar, S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar, S. J. Genet... Scholar). cause two cause and one results from a Transmission electron micrographs of fibrils in four cases are by the of fibril and in diameter. null of have been as a cause of a proportion of EDS Steinmann B. Am. J. Hum. Genet... Am. J. Hum. Genet... Scholar). Transmission electron micrographs in one showed larger and fibrils Am. J. Hum. Genet... Scholar). In the present have and a type V collagen mutation a null allele. of type V collagen is both collagen and salt precipitation of proband secreted collagen of the α1(V)2α2(V) chain composition of type V collagen even in the context of α1(V) haploinsufficiency. dermal fibrils have greater heterogeneity of diameter than in the control. The of for the genotype/phenotype in EDS are a type V collagen defect in a with type of Ehlers-Danlos Syndrome. collagen exons an of the helical R. B. Proc. Natl. Acad. Sci. U. S. A... defects that in the of an exon the reading frame and cause the of a abnormal α the G defect in an α1(V) exon 14 acceptor site in the of a cryptic acceptor site −100 in intron The cryptic acceptor had the than the usual of α1(V) exon 14 and fibrillar collagen splice acceptor In there were a of in the 3′-end of intron 13 in which the from to of the mutant transcript by with an at the 3′-end of intron 13 with in exon or 13 revealed additional The of the −100 site from the of a or in the The site at with or was the candidate and have been in α1(V) chain be from this transcript with a the insertion shifts the collagen reading frame +1 and causes the of a stop codon in exon 17. α chain from the mutant transcript be into V the and region in the of α J. Scholar). In the α1(V) exon 14 the between the and the helical region Scholar). transcripts α helical a and be only a amount of even the chain be because the mutant transcript is to nonsense-mediated decay and is present in the fibroblasts at only 8% the level of normal These features to a null allele at the is in the of type V collagen secreted by the fibroblasts of the proband. the of secreted collagen is to of type I collagen as the proband as much type V collagen as the control. that the fibrils of the proband must be with a type I collagen Selective salt precipitation was to the of the type V trimer secreted in the context of α1(V) has the same of α1(V) and have been that the chain was a of the tissue In only α1(V) was Miller Scholar). In Miller and skin A. Biochem. the two were present in a In bone, the trimer was of a of and the chain Tissue Scholar). Furthermore, the chain is in fibroblasts J. Biol. Chem... Scholar). of α1(V) the of the type V In the collagen secreted by the type V collagen forms were at the and the α1(V) and were present in a is that trimer composition is tissue from an the type V collagen in proband is secreted in a amount and with the usual α chain the of this mutant for matrix and for must from V than a is a for a proportion of EDS I and of which this is the in and Am. J. Hum. Genet... α1(V) from cases of EDS I or In were to or or to stop Steinmann B. Am. J. Hum. Genet... the of EDS patients for of patients were for at one length and of had only one allele in The of two of patients that at of EDS I or patients have mutations in which results in haploinsufficiency. Furthermore, of mutations in of mutant transcript by nonsense-mediated decay is one type of which transcripts with are by a D. R. Scholar). are to have a greater decay in the transcript J. and the codon in this that must also be because of the cases of EDS with are in the The context of the codon is also to be the of one or by A. 1993; Scholar). are 14 and of the stop codon in the mutant transcript of this null mutation with V structural defects genotype/phenotype of V structural of α1(V) and three structural of have been Miller Genet... Scholar, Hum. Genet... Scholar, S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar, S. J. Genet... Scholar). are and the are or and a The of patients with V structural the range of EDS I II. The family has to EDS II. The cases in the by Am. J. Hum. Genet... and Steinmann B. Am. J. Hum. Genet... the EDS to type V appears to have a than a null allele of type I collagen. type I collagen, null of are for the form of osteogenesis whereas structural defects of type I collagen cause the severe to and IV of osteogenesis In EDS, the of to be similar to those of structural between the genotype/phenotype of I and V collagen in in type I collagen null a of fibrils not have a tissue and causes osteogenesis In because of the role of type V collagen in fibril both and structural defects of type V collagen cause of fibril which are to the of the skin and The four cases of EDS with V structural defects for which electron of fibrils has been defects of exon α1(V) exon α1(V) exon and an α1(V) substitution S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar). the same of fibril and in diameter. It is that this is also the fibril seen in this in which the defect causes because a in fibril diameter have been that the of fibril in is not the of V and I collagen. there is a in the of type V collagen fibrils with fibrils normal in and In of the of null mutations in and fibril a for to the of a type V collagen defect in a with type of Ehlers-Danlos Syndrome. collagen exons an of the helical R. B. Proc. Natl. Acad. Sci. U. S. A... defects that in the of an exon the reading frame and cause the of a abnormal α the G defect in an α1(V) exon 14 acceptor site in the of a cryptic acceptor site −100 in intron The cryptic acceptor had the than the usual of α1(V) exon 14 and fibrillar collagen splice acceptor In there were a of in the 3′-end of intron 13 in which the from to of the mutant transcript by with an at the 3′-end of intron 13 with in exon or 13 revealed additional The of the −100 site from the of a or in the The site at with or was the candidate and have been in α1(V) chain be from this transcript with a the insertion shifts the collagen reading frame +1 and causes the of a stop codon in exon 17. α chain from the mutant transcript be into V the and region in the of α J. Scholar). In the α1(V) exon 14 the between the and the helical region Scholar). transcripts α helical a and be only a amount of even the chain be because the mutant transcript is to nonsense-mediated decay and is present in the fibroblasts at only 8% the level of normal These features to a null allele at the is in the of type V collagen secreted by the fibroblasts of the proband. the of secreted collagen is to of type I collagen as the proband as much type V collagen as the control. that the fibrils of the proband must be with a type I collagen Selective salt precipitation was to the of the type V trimer secreted in the context of α1(V) has the same of α1(V) and have been that the chain was a of the tissue In only α1(V) was Miller Scholar). In Miller and skin A. Biochem. the two were present in a In bone, the trimer was of a of and the chain Tissue Scholar). Furthermore, the chain is in fibroblasts J. Biol. Chem... Scholar). of α1(V) the of the type V In the collagen secreted by the type V collagen forms were at the and the α1(V) and were present in a is that trimer composition is tissue from an the type V collagen in proband is secreted in a amount and with the usual α chain the of this mutant for matrix and for must from V than a is a for a proportion of EDS I and of which this is the in and Am. J. Hum. Genet... α1(V) from cases of EDS I or In were to or or to stop Steinmann B. Am. J. Hum. Genet... the of EDS patients for of patients were for at one length and of had only one allele in The of two of patients that at of EDS I or patients have mutations in which results in haploinsufficiency. Furthermore, of mutations in of mutant transcript by nonsense-mediated decay is one type of which transcripts with are by a D. R. Scholar). are to have a greater decay in the transcript J. and the codon in this that must also be because of the cases of EDS with are in the The context of the codon is also to be the of one or by A. 1993; Scholar). are 14 and of the stop codon in the mutant transcript of this null mutation with V structural defects genotype/phenotype of V structural of α1(V) and three structural of have been Miller Genet... Scholar, Hum. Genet... Scholar, S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar, S. J. Genet... Scholar). are and the are or and a The of patients with V structural the range of EDS I II. The family has to EDS II. The cases in the by Am. J. Hum. Genet... and Steinmann B. Am. J. Hum. Genet... the EDS to type V appears to have a than a null allele of type I collagen. type I collagen, null of are for the form of osteogenesis whereas structural defects of type I collagen cause the severe to and IV of osteogenesis In EDS, the of to be similar to those of structural between the genotype/phenotype of I and V collagen in in type I collagen null a of fibrils not have a tissue and causes osteogenesis In because of the role of type V collagen in fibril both and structural defects of type V collagen cause of fibril which are to the of the skin and The four cases of EDS with V structural defects for which electron of fibrils has been defects of exon α1(V) exon α1(V) exon and an α1(V) substitution S. J. Genet... Scholar, A. Am. J. Hum. Genet... Scholar, Am. J. Hum. Genet... Scholar, J. Hum. Genet... Scholar). the same of fibril and in diameter. It is that this is also the fibril seen in this in which the defect causes because a in fibril diameter have been that the of fibril in is not the of V and I collagen. there is a in the of type V collagen fibrils with fibrils normal in and In of the of null mutations in and fibril a for to the of We the of the family in this especially for and We B. of and D. for the two of the Ehlers-Danlos Syndrome collagen reverse transcriptase-polymerase chain chain nucleotide

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